The growth or atrophy of mammalian tissues is determined by the relative rates of protein degradation and protein synthesis. Despite the imporant role of protein degradation in the regulation of cellular protein content, it is unclear which enzymes are responsible for the intracellular degradation of proteins or how proteolysis is regulated. The long term objectives of the proposed research are: to identify the enzymes which catalyze intracellular protein degradation in mammalian tissues, to understand the mechanism which regulate the activities of these enzymes, and to determine how these proteases function in the regulation of protein degradation in normal physiologic and pathologic conditions. This study examines three cytoplasmic proteases of rat liver: two calcium-dependent proteases and an unusual high-molecular weight protease which is stimulated by ATP. Because calcium and ATP are known to modulate overall rates of protein degradation in intact cells, these proteases and the mechanisms by which their activities are regulated may be particularly important in intracellular proteolysis. This project will define the physical and catalytic properties of these enzymes and study the factors and mechanisms which regulate their activities. The calcium-dependent proteases have indistinguishable heterodimer subunit compositions but one protease is half-maximally activated at 250 MuM Ca2+ while the other is half-maximally activated at 1 MuM Ca2+. The physical relationship between the two enzymes will be determined by chemical and immunologic methods to demonstrate whether the enzymes represent distinct proteins or whether they are interconverted forms of the same protein. The functions of each subunit will also be assessed. The activity of each protease appears to be regulated by two specific regulatory proteins, and by limited proteolysis. The influence of each of these factors on various catalytic properties of each protease will be studied in detail and correlated with protease structure. The high molecular weight protease will be purified by affinity chromatography. Its physical properties such as subunit composition will be determined. The mechanism of ATP-stimulation on protease activity will be determined. The catalytic action of each of the three proteases will be characterized against several proteins which are potential physiologic substrates. The specificity and extent of hydrolysis of the substrates by each protease will be determined and compared.